Interferon gamma decreases intestinal epithelial aquaporin 3 expression through downregulation of constitutive transcription

Michael A. Peplowski, Michael Dicay, Cristiane H. Baggio, Filip Wysokinski, Bernard Renaux, Morley D. Hollenberg, David Proud, Wallace K. MacNaughton

Aquaporin (AQP) 3 expression is altered in inflammatory bowel diseases, although the exact mechanisms regulating AQP abundance are unclear. Although interferon gamma (IFNγ) is centrally involved in intestinal inflammation, the effect of this cytokine on AQP3 expression remains unknown. HT-29 human colonic epithelial cells were treated with IFNγ to assess AQP3 mRNA expression by real-time RT-PCR and functional protein expression through the uptake of radiolabelled glycerol. Transient knockdown of signal transducer and activator of transcription 1 (STAT1), STAT3, Sp1, and Sp3 were performed to determine the involvement of these transcription factors in the IFNγ-induced signalling cascade. AQP3 promoter regions involved in the response to IFNγ were assessed using a luciferase reporter system. Likewise, enteroids derived from human colonic biopsies were also treated with IFNγ to assess for changes in AQP3 mRNA expression. IFNγ decreased AQP3 mRNA expression in HT-29 cells in a time- and concentration-dependent manner and reduced functional AQP3 protein expression (decreased 3H-labelled glycerol uptake). IFNγ also reduced AQP3 expression in enteroids derived from human colonic biopsies. Knockdown of STAT1 partially prevented the IFNγ-induced downregulation of AQP3 expression, whereas STAT3 and Sp3 knockdowns resulted in increased baseline expression of AQP3 but did not alter IFNγ-induced downregulation. Constitutive transcription of AQP3 is downregulated by IFNγ as demonstrated using the luciferase reporter system, with Sp3 bound to the AQP3 promoter as shown by chromatin immunoprecipitation. AQP3 constitutive transcription in intestinal epithelial cells is downregulated by IFNγ. This response requires STAT1 that is postulated to drive the downregulation of AQP3 expression through increased acetylation or decreased deacetylation the AQP3 promoter, ultimately resulting in decreased constitutive transcription of AQP3. • IFNγ suppresses the expression of AQP3 in intestinal epithelial cells. • Proximal AQP3 promoter elements are sufficient to drive constitutive expression and mediate the IFNγ-induced downregulation of AQP3 mRNA expression. • IFNγ-induced suppression of AQP3 is dependent upon STAT1 expression, but not STAT3, Sp1, or Sp3.